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Targeting mammalian organelles with internalizing phage (iPhage) libraries

Nature Protocols volume 8, pages 1916–1939 (2013)Cite this article

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Abstract

Techniques that are largely used for protein interaction studies and the discovery of intracellular receptors, such as affinity-capture complex purification and the yeast two-hybrid system, may produce inaccurate data sets owing to protein insolubility, transient or weak protein interactions or irrelevant intracellular context. A versatile tool for overcoming these limitations, as well as for potentially creating vaccines and engineering peptides and antibodies as targeted diagnostic and therapeutic agents, is the phage-display technique. We have recently developed a new technology for screening internalizing phage (iPhage) vectors and libraries using a ligand/receptor-independent mechanism to penetrate eukaryotic cells. iPhage particles provide a unique discovery platform for combinatorial intracellular targeting of organelle ligands along with their corresponding receptors and for fingerprinting functional protein domains in living cells. Here we explain the design, cloning, construction and production of iPhage-based vectors and libraries, along with basic ligand-receptor identification and validation methodologies for organelle receptors. An iPhage library screening can be performed in ∼8 weeks.

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Figure 1: Flowchart of the iPhage combinatorial library technology.
Figure 2: The iPhage vector and library cloning strategy.
Figure 3: An overview of phage vector (f88-4, fUSE5 and iPhage) purification by CsCl.
Figure 4: A systematic approach for affinity chromatography and receptor identification based on the iPhage technology.
Figure 5: An overview of differential centrifugation and subcellular fractionation quality analysis.
Figure 6: iPhage organelle-targeting properties.
Figure 7: Combinatorial screening of peptides targeting subcellular compartments and the identification of candidate receptors by affinity chromatography and MS/MS analysis.

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Acknowledgements

This work was supported by grants from the US National Institutes of Health, the US Department of Defense, and by awards from The University of Texas M.D. Anderson Cancer Center Trust, the Marcus Foundation, AngelWorks and the Gillson-Longenbaugh Foundation (all to W.A. and R.P.). R.R. received support from the Odyssey Scholar Program at the University of Texas M.D. Anderson Cancer Center.

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Author notes

  1. Roberto Rangel & Liliana Guzman-Rojas

    Present address: Present address: Cancer Research Program, Houston Methodist Research Institute, Houston, Texas, USA.,

  2. Roberto Rangel, Andrey S Dobroff and Liliana Guzman-Rojas: These authors contributed equally to this work.

  3. Renata Pasqualini and Wadih Arap: These authors jointly directed this work.

Authors and Affiliations

  1. David H. Koch Center, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA

    Roberto Rangel, Andrey S Dobroff, Liliana Guzman-Rojas, Carolina C Salmeron, Renata Pasqualini & Wadih Arap

  2. Department of Biomedical Engineering, Wayne State University, Detroit, Michigan, USA

    Juri G Gelovani

  3. Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA

    Richard L Sidman

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Contributions

R.R., A.S.D., L.G.-R., J.G.G., R.L.S., R.P. and W.A. designed experiments. R.R., A.S.D., L.G.-R. and C.C.S. performed the experiments. R.R., A.S.D., L.G.-R., C.C.S., J.G.G., R.L.S., R.P. and W.A. analyzed data. R.R., A.S.D., L.G.-R., R.L.S., R.P. and W.A. wrote the manuscript. R.P. and W.A. supervised the project.

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Correspondence to Renata Pasqualini or Wadih Arap.

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Competing interests

The University of Texas M.D. Anderson Cancer Center (UTMDACC), along with its researchers (R.R., L.G.-R., R.P., W.A.), has filed patents on the technology and intellectual property reported here. If licensing or commercialization occurs, the researchers are entitled to standard royalties. R.P. and W.A. have equity in AAVP Biosystems. UTMDACC manages the terms of these arrangements in accordance with its established institutional conflict of interest policies.

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Rangel, R., Dobroff, A., Guzman-Rojas, L. et al. Targeting mammalian organelles with internalizing phage (iPhage) libraries. Nat Protoc 8, 1916–1939 (2013). https://doi.org/10.1038/nprot.2013.119

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